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Background The recent discovery of a mecA homologue (mecA(LGA251)) with a high level of variability between the two gene variants suggested that Staphylococcus aureus harbouring mecA(LGA251) could be wrongly identified as methicillin-susceptible S. aureus (MSSA), in the absence of antimicrobial susceptibility testing. Methods In this context we designed a real-time quadruplex PCR assay to distinguish unequivocally between mecA and mecA(LGA251), alongside the nuc gene (a species-specific marker) and detection of the lukS-PV gene [encoding the Panton-Valentine leucocidin (PVL) toxin]. Results and discussion The assay was validated using a collection of (i) PVL-positive and PVL-negative MSSA and methicillin-resistant S. aureus (MRSA) and (ii) known MRSA harbouring mecA(LGA251) from the UK, Denmark and France. When applied to a retrospective collection of oxacillin-non-susceptible, mecA-negative human isolates, three were found to encode mecA(LGA251), including one from blood, representing the first hitherto recognized case of bacteraemia due to S. aureus possessing the mecA(LGA251) in England. Finally, the assay was introduced into the routine Staphylococcus Reference Unit (HPA Microbiology Services, London, UK) workflow in August 2011, and, during the first 5 months of use, 10 isolates harbouring the mecA homologue were identified out of 2263 S. aureus tested, suggesting a low but continuous circulation within the human population in England.

Objectives: To analyse the sequence diversity of the tetracycline resistance gene tet(M) in Staphylococcus aureus from humans and animals and to determine mobile elements associated with tet(M) in S. aureus. Methods: In total, 205 tetracycline-resistant isolates were screened for tet(M) by PCR. tet(M) genes were sequenced and compared with tet(M) deposited in GenBank. Based on phylogenetic analysis isolates were screened for Tn916- and Tn5801-like xislint genes, and transposons were confirmed by linking PCR. spa typing was performed and selected isolates were used as donors in a filter mating experiment. Results: Forty-one isolates (21.3%, 60.7%, 2.6% and 4.4% of the human, pig, poultry and cattle isolates, respectively) were tet(M) positive. tet(M) was located on Tn5801-like and Tn916-like transposons in humans and on a specific Tn916-like element in animals. Human isolates were of different spa types (t034, t008, t037, t051, t065, t078, t318 and t964) corresponding to different clonal complexes (CC398, CC8, CC25 and CC30). Animal isolates were of spa type t034, t011 or t0571 corresponding to CC398. tet(M) sequence types correlated with CC types. Tn916-like and Tn5801-like (Tn6014) transposons were able to transfer to S. aureus recipients. Conclusion: S. aureus of human origin contained diverse tet(M) located on Tn916- and Tn5801-like (Tn6014) transposons, and S. aureus of animal origin contained Tn916-like tet(M) genes. This suggests that conjugative transposition plays an important role in the evolution and horizontal spread of tet(M) in S. aureus. This is the first study showing horizontal transfer of Tn5801 (Tn6014).

Aerococcus urinae is a newcomer in clinical and microbiological practice, causing urinary tract infections, bacteraemia/septicaemia and/or endocarditis. This study presents for the first time an evaluation of the activity of a representative panel of antibiotics against a large number of A. urinae isolates. The in vitro susceptibilities (MICs) of 56 isolates of A. urinae to 14 antibiotics were determined by agar dilution. In general, A. urinae isolates showed little inter-isolate variability, and had low MICs of penicillin, amoxicillin, piperacillin, cefepime, vancomycin and rifampicin. High-level aminoglycoside resistance was not found for any of the isolates. Moderate to good activity was seen with quinolones, erythromycin and tetracycline. Isolates from two patients with endocarditis were studied with time-kill curves for penicillin, gentamicin and vancomycin. Penicillin and vancomycin alone exhibited slow or no bactericidal activity against the two strains. When combining either penicillin or vancomycin with gentamicin, rapid bactericidal activity was obtained for both strains with both combinations. The treatment options for A. urinae seem to include penicillins for less severe cases. In severe cases, i.e. endocarditis, the time-kill investigations suggest a beneficial effect of combination with gentamicin. In the penicillin-allergic patient vancomycin in combination with gentamicin represents the most obvious alternative.

A new 3-h hybridization assay for detection of the staphylococcal mecA gene and the Staphylococcus aureus nuclease gene was evaluated by comparing the assay with existing genotypic and phenotypic methods. A total of 275 S. aureus strains were tested, including 257 epidemiologically unrelated strains (135 mecA-positive and 122 mecA-negative; collection I), and 18 strains with known borderline resistance to methicillin (collection II). Complete agreement was obtained for both collections when comparing the new assay with genotypic methods. We further evaluated a range of phenotypic susceptibility methods recommended in Europe and/or USA using the presence of the mecA gene as the defining standard. For collection I a high degree of agreement was found for both Etests (256 strains) and the oxacillin screen plate test (255 strains); the degree of agreement was lower for agar dilution methicillin (250 strains) and oxacillin 1 microg discs (239 strains). For the borderline strains a high degree of agreement was only obtained by the oxacillin screen plate test (17 of 18 strains). The other tests were less accurate, in the following order: agar dilution methicillin, Etest methicillin, Etest oxacillin and oxacillin discs with disagreement for four, five, nine and 13 strains, respectively. In conclusion, the new hybridization assay is a rapid and exact method for detecting the mecA gene and the S. aureus nuclease gene. This study confirms that phenotypic tests for methicillin resistance in S. aureus strains creates both false-susceptible and false-resistant results, especially for borderline resistant strains.

Received 22 February 2013; returned 22 March 2013; revised 2 April 2013; accepted 7 April 2013 Objectives: Chlorhexidine is used as a disinfectant to prevent surgical infections. Recently, studies have indicated thatchlorhexidineusagehasselectedmethicillin-resistantStaphylococcusaureusstrainsthataretoleranttochlorhexidine and that this may be related to the presence of the qacA/B-encoded efflux pumps. Here, we evaluated if high-level exposure to chlorhexidine selects for tolerant colonizing Staphylococcus epidermidisand we addressed the consequences of long-term exposure to chlorhexidine. Methods:ChlorhexidinesusceptibilityandcarriageofqacA/BwasdeterminedforcolonizingS.epidermidisisolated fromscrubnursesheavilyexposedtochlorhexidineandwerecomparedwithisolatesfromnon-usersofchlorhexidine hand rubs.S. epidermidisblood isolatesfrom the1960s, before thewider introductionof chlorhexidine to the market, were also tested and compared with recently collected S. epidermidis blood isolates. Results: There was no correlation between the use of chlorhexidine in scrub nurses and the presence of qacA/B genes in S. epidermidis isolates or increased MICs/MBCs of chlorhexidine for S. epidermidis isolates. While 55% of current blood isolates harboured the qacA/B genes, none of the 33 historical S. epidermidis isolates did, although their MICs and MBCs of chlorhexidine were comparable to those for current isolates. Conclusions: Chlorhexidine used as a hand rub does not select for S. epidermidis isolates with increased MICs or MBCsofchlorhexidine.However,theabsenceofqacA/BgenesinS.epidermidisisolatesobtainedinthe1960ssuggeststhatlong-termuseofbiocideslikechlorhexidineorrelatedcompoundsmayselectforthepresenceofqacA/B genes.

The EVIGENE MRSA Detection Kit was evaluated on coagulase-negative staphylococci (CoNS) from agar plates and on staphylococci directly from positive spiked blood cultures. For the CoNS study, a total of 242 isolates were tested, and of these 237 gave valid test results. For the 237 valid tests, all gave correct mecA classification. For the blood culture procedure, a collection of 51 mecA-positive Staphylococcus aureus, 21 mecA-negative S. aureus, 31 mecA-positive CoNS and 28 mecA-negative CoNS were used for the simulated blood cultures. For the S. aureus strains, all gave valid test results and correct mecA classification. One of the MRSA isolates gave a very faint nuc signal, and another four isolates gave results close to the cut-off of the kit; however, these were still clearly positive when read by the naked eye. For the CoNS isolates, 51 of the 59 strains gave valid results. All of these 51 strains gave correct mecA status. Thus the EVIGENE MRSA Detection Kit can provide fast and accurate determination of methicillin resistance in CoNS. This preliminary study of the blood culture procedure indicates that it is possible to achieve determination of methicillin resistance in staphylococci 8 h after positivity of the blood culture, making same-day detection of methicillin resistance possible.

LEO Pharma, Industriparken 55, DK-2750 Ballerup, Denmark; Statens Serum Institut, Artillerivej 5, DK-2300 Copenhagen, Denmark; Service de Bacteriologie-Hygiene, CHU Pitie-Salpetriere, Paris, France; Hopital Robert Debre, Service Dermatologie, Avenue de General Koening, F-51092 Reims Cedex, France; Department of Clinical Microbiology, Central Pathology Laboratory, St James’s Hospital, Dublin 8, Ireland; Department of Clinical Microbiology, Leeds General Infirmary, Great George Street, Leeds, West Yorkshire LS1 3EX, UK